Healthcare-associated infections (HCAIs), or nosocomial infections, are those that are acquired by a patient during the course of receiving treatment within a healthcare setting. HCAIs can affect any part of the body, including the urinary system, the respiratory system, the skin, surgical wounds, the gastrointestinal system and even the bloodstream. Many such infections may arise from the presence of micro-organisms present on the body of the patient; however, they may also be caused by micro-organisms originating from another patient or from micro-organisms transmitted from the hospital environment due to poor hygiene.
HCAIs are amongst the major causes of death and increased morbidity in hospitalised patients. Each year, at least 2,000,000 patients in the USA and over 320,000 patients in the UK acquire one or more HCAIs during their stay in hospital. It is predicted that 1 in 4 patients in intensive care worldwide will acquire an infection during their treatment; this estimate is double in less developed countries. It has been reported that at any one time more than 1.4 million people worldwide are suffering from an HCAI. As a consequence, it is predicted patients spend an average of 2.5 times longer in hospital. In addition to a direct consequence on patient safety, HCAIs also constitute a significant financial burden on healthcare systems. In the USA, the risk of HCAIs has risen steadily over the last decade with accompanying costs estimated at US$ 4.5-5.7 billion a year. Similarly in the UK, HCAIs are estimated to cost the NHS £1 billion a year.
The micro-organisms giving rise to HCAIs are numerous, and may be in the form of any number of pathogens such as bacteria, virus, fungus, parasites or prions. The most commonly known nosocomial pathogens include methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile (C. difficile) and Escherichia coli (E. coli).
C. difficile is a species of Gram-positive bacteria of the genus Clostridium. C. difficile is a commensal anaerobic bacterium of the human intestine present in approximately 2-5% of the population. However, an imbalance of gut bacterial load and infection with C. difficile can result is severe diarrhoea and intestinal dysfunction. The disease spectrum caused by C. difficile infection (CDI) ranges from mild self-limited illness to severe life-threatening conditions. Other example species of Clostridium causing human disease include C. perfringens, C. tetani, C. botulinium, C. sordeffii and C. difficile. Clostridial species are associated with diverse human diseases including tetanus, gas gangrene, botulism and pseudomembraneous colitis and can be a causative agent in food poisoning.
The pathogenesis of C. difficile has been attributed to various described putative virulence factors. The most widely described are the clostridial toxins designated toxin A (TcdA) and toxin B (TcdB), both of which are monoglucosyltransferases that are cytotoxic, enterotoxic, and pro-inflammatory. They belong to the large clostridial cytotoxin (LCT) family, and share approximately 66% amino acid homology with each other. The toxins are thought to target and inactivate the Rho-Rac family of GTPases in the host epithelial cells of the gut. This has been shown to result in actin depolymerisation by a mechanism correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins. This eventually results in massive fluid secretion, acute inflammation, and necrosis of the colonic mucosa.
C. difficile is identified as the most common cause of antibiotic associated diarrhoea. Since 2000, there has been a dramatic increase in the rates and severity of CDI particularly in North America and Europe. The primary risk factor for the development of CDI is the use of antibiotics disrupting the normal enteric bacterial flora enabling an overgrowth of ingested or endogenous C. difficile. However, the population at risk of suffering from CDI includes not only patients on antimicrobial and other therapies that can alter the balance of the gut flora, but also the immune-compromised (such as a consequence of disease or medical treatment) and the elderly. Other increased risk factors for CDI include length of hospital stay, use of feeding tubes, mechanical ventilation, invasive cannulae or catheters, and underlying co-morbidity. Accounts of relapse or re-infection of C. difficile in susceptible individuals is also documented with 15-35% of patients suffering relapse within the first 2 months post treatment.
Additionally, the incidence of CDI is further increased due to the sporulating nature of C. difficile. High levels of bacterial spores are present in hospital environments. Indeed, it has recently been shown that many standard hospital cleaning agents are ineffective at eradicating Clostridial spores from the environment, resulting in ineffective disease control.
The course of treatment of CDI can vary depending upon the stage and severity of the disease. For example, in early diagnosed or mild to moderate infections caused as a consequence of antibiotic administration, ceasing the treatment can re-establish the natural gut flora. However, in more severe cases, or recurring cases, vancomycin can be administered. Other treatment methods, particularly in the cases of multiple relapse, include re-establishment of the gut flora, for example, through use of probiotics such as Saccharomyces boulardi or Lactobacillus acidophilus, or by faecal bacteriotherapy (stool transplantation from an uninfected individual). However, two of the most difficult challenges for CDI treatment are the management of multiple recurrences and the management of fulminant or severe complicated CDI, in which cases if all existing treatment regimens fail, surgical removal of the colon can be the only remaining life-saving measure (Rupnik et al., 2009).
Prior to the production and release of toxinogenic compounds, the ingested spores of C. difficile remain dormant, forming non-reproductive structures in response to environmental stress. Once environmental conditions become favourable, the spores germinate and the bacteria proliferate into vegetative cells, which colonize the gastrointestinal (GI) tract of susceptible individuals. Bacterial spores, however, are extremely tolerant to many agents and environmental conditions including radiation, desiccation, temperature, starvation and chemical agents. This natural tolerance to chemical agents allows spores to persistent for many months in key environments such as hospitals or other healthcare centres.
Furthermore, many standard treatment methods, drugs, or antibacterial agents commonly used to treat bacterial infections have been associated with an increased risk of C. difficile colonisation and subsequent development of CDI. For example, typical antibacterial agents used to treat Helicobacterpylori infections are a combination of metronidazole, amoxicillin, levofloxacin or clarithromycin, all of which have strongly been associated with the development of CDI. Additionally, vancomycin used for the treatment of CDI is of concern due to its bacteriostatic action, relatively high cost and the possible development of resistance in C. difficile strains (and other bacteria). In fact, nearly every antibiotic has been associated with subsequent CDI, but some carry a higher risk than others (Rupnik et al., 2009). Current therapies are therefore extremely limited in the treatment or control of C. difficile related infections particularly in view of the fact nearly all antibiotic classes are associated with causing or exacerbating the disease.
Consequently, there is an increasing need to develop new and efficacious agents or treatment regimens to reduce the incidence and likelihood of suffering from CDI, and other similar diseases associated with spore forming bacteria. Many existing therapies to treat or reduce the incidence of CDI and C. difficile spread either attempt to re-establish the native gut microorganism population, reduce the levels of C. difficile toxins or stimulate the immune system.
The exact mechanism by which colonization of the gut by C. difficile occurs, and therefore how the organism establishes itself in the gut to lead to CDI, remains unclear. Colonisation is thought to enable the organism to penetrate the mucus layer and attach to the underlying colonic epithelial cells, thereby facilitating the delivery of toxins to host cell receptors. Surface attachment is a property of many bacterial species, which permits colonization of specific niches. For many pathogenic bacteria, formation of biofilms (containing an extracellular polymeric substance EPS matrix) is linked to colonization, maintenance and disease. Biofilms are defined as polymicrobial aggregates attached to each other and/or to surfaces. These aggregates are referred to by several terms; films, mats, flocs, sludge or biofilms. Many pathogenic bacteria form biofilms in response to a diverse array of environmental cues, for self-preservation, evasion of the host immune response, mechanical integrity and preservation of nutrients, or colonization of a particular niche. As well as providing a protective environment from external influences such as, host immune response, desiccation, biocides and some antibiotics, biofilms can also provide a nutrient source and promotes recycling of lysed cells. It has been shown that bacterial surface structures such as flagella, pili and fimbriae can stabilize the biofilm.
In some species the extracellular polymeric substance (EPS) matrix accounts for as much as 90% of the biofilm biomass, with the remainder comprising bacteria. The EPS matrix provides the scaffold by which bacteria adhere to each other and to surfaces. The composition and architecture of the EPS is species and even strain dependent but the majority are comprised of polysaccharides, nucleic acids, lipids and proteins, which are secreted by biofilm producing bacteria. Furthermore, the composition of an EPS can vary greatly between biofilms of the same species and is influenced by many factors, including the microorganisms present, the shear forces experienced, the temperature and the availability of nutrients. The identification of a biofilm and its components depends on the isolation and culture methods used, particularly as the composition can vary, and therefore different experimental methods are required to isolate different biofilms. Consequently, the nature and identification of biofilms has to be specific for each type of biofilm under investigation.
Currently, there exists uncertainty as to whether Clostridium species, and in particular C. difficile, produces a biofilm. Biofilms have been found to be involved in a wide variety of microbial infections in the body, it has been suggested that they are involved in 80% of all infections, with common examples including urinary tract infections, catheter infections, middle-ear infections, formation of dental plaque, endocarditis, and infections symptomatic of cystic fibrosis. Given these facts, researchers consider C. difficile may produce a biofilm to promote colonisation. However, reports in the art are conflicting since Reynolds et al, 2010 state C. difficile does not exhibit biofilm formation, whereas Shirtliff et al 2012 believe it does (http://www.dental.umaryland.edu/dentaldepts/micropath/shirtliff_lab_projects_clostridium.html).
Therefore, it is generally acknowledged that is unknown to what extent, if any, adhesion and biofilm production are involved in the pathogenesis of C. difficile (Rupnik et al., 2009).
However, our investigations have revealed conclusive evidence that a variety of clinical isolates of C. difficile form biofilms, in both rich and non-rich media. Furthermore, we have shown for the first time that that both the super-structure and sub-structures of these biofilms differ depending on environmental conditions, such as nutrient availability and growth parameters. This hitherto unknown characteristic may contribute to the pathogenicity of the bacteria, providing protection against immune responses, and permitting adhesion and colonisation of the gut leading to infection. Furthermore, uniquely, we have found that the biofilms comprise a substantial number of live bacteria, which may serve to provide a reservoir of C. difficile for re-colonising the host following treatment and so may account for the significant relapse incidence associated with C. difficile infection.
Moreover, our further analysis of the C. difficile biofilm revealed its primary component is nucleic acid which we discovered, upon treatment with deoxyribonuclease, is degraded effectively in a dose dependent manner. Additionally, we also discovered that pre-treatment of bacterial culture vessels with deoxyribonuclease inhibited the formation of a C. difficile biofilm. Therefore, the use of deoxyribonuclease against CDI represents a potential new avenue for therapeutic intervention in the treatment of CDI, potentially reducing the pathogenicity of the bacteria and its ability to colonise the host, in addition to increasing its susceptibility to host immune defence mechanisms and sensitivity to other existing antibacterial agents. Consequently, this has important implications in treating CDI, reducing the incidence of disease spread, and relieving the burden currently shouldered by healthcare organisations.